Detection of antibodies against chamydia trachomatis PGP3 antigen in patient sera by enzyme-linked immunoassay

ABSTRACT

A new recombinant form of the plasmid-encoded protein pgp3 from  C. trachomatis,  serotype D, was purified by ion exchange column chromatography and shown to be suitable for quantitative immunoassay on clinical samples in an ELISA format.

RELATED APPLICATIONS

The present application is a division of U.S. Ser. No. 10/365,602, filedFeb. 13, 2003 which is a continuation of U.S. Ser. No. 08/462,151 filedJun. 5, 1995, which is a division of U.S. Ser. No. 08/229,980 filed Apr.19, 1994, all of which are incorporated herein in their entireties.

FIELD OF THE INVENTION

A new recombinant form of the plasmid-encoded protein pgp3 from C.trachomatis, serotype D, was purified by ion exchange columnchromatography and shown to be suitable for quantitative immunoassay onclinical samples in an ELISA format. Since initial attempts ofdeveloping a similar assay with a SDS-denatured pgp3-fusion proteinfailed, the results suggest that for anti-pgp3 antibody detection,antigen conformation is important.

BACKGROUND OF THE INVENTION

The function of the 7.5 kb plasmid, pCT, of Chlamydia trachomatis isstill unknown. However the fact that this DNA element appears to bestrongly conserved in C. trachomatis (both in terms of its presence inessentially all isolates and in terms of its genetic structure) suggeststhat pCT may provide some, important, and perhaps advantageous, functionto the chlamydial cell during its natural host infection. Recently, anopen reading frame of pCT (ORF3; Comanducci et al., Plasmid, 23:149-154,1990) was expressed in E. coli as a recombinant fusion protein(Comanducci et al., J. Gen. Microbiology, 134:1083-1092, 1993). Theexpression system used added a 11-kDa N-terminal polypeptide of theMS2-bacteriophage polymerase to the 28-kDa polypeptide (pgp3) encoded byORF3. The resulting 39-kDa product was used to show that pgp3 epitopescan be recognized on Western blots by antibodies present in sera frompatients with chlamydial infections, but not in control sera fromhealthy donors. Following this observation, we subsequently tried todevelop a serological test more suitable than the immunoblottingtechnique for obtaining reproducible and quantitative data from largenumbers of clinical samples. We now report that an enzyme-linkedimmunoassay based on a new recombinant form of the pgp3 protein, can beused for assessing the prevalence of pgp3 antibodies in people with C.trachomatis infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Reaction of human sera with plastic bound recombinant pgp3protein. Results obtained with sera diluted from 200 to 6800 folds areshown. ODs are average values obtained from duplicate samples. C.trachoratis MIF titres of the sera (top to bottom) were 1:256; 1:128;1:128; 1:32; zero. Sera C, A, and 3 were from women with salpingitis;the serum B was from a male with isolation-positive chlamydialurethritis; serum 13 was from a healthy blood donor.

DETAILED DESCRIPTION OF THE INVENTION

Initial attempts of developing an ELISA test for human sera using thepreviously described (Comanducci et al, 1993) 39-kDa pgp3 fusionprotein, purified by electrophoresis on SDS-acrylamide gels, gaveinconsistent and unsatisfactory results. One of the problems was theobservation that, in some patient sera, antibody reaction with minorcontaminants from E. coli was stronger than the reaction with thedenatured fusion protein. Situations like this can be adequatelyresolved by the Western blot technique, where signals from differentantigens can be separately monitored, but they become a serious problemin ELISA. Therefore, we attempted to improve both the quality of therecombinant antigen and purification procedures. Considering thatpatients reacting against pgp3 during a chlamydial infection will alsomake antibodies against conformational epitopes (which are not expectedto give an effective interaction with the largely denatured antigensusually present on Western blots), we decided to seek expression in E.coli of the pgp3 antigen with its native amino acid sequence and adopt apurification procedure which avoided, as much as possible, denaturingsteps. This was obtained by using the pT7.7 expression system in E. coliBL21 cells, and column chromatography of cell extracts on a Mono-Qsupport. The new recombinant pgp3 antigen was initially tested for itsability to bind to microtiter plate wells in a variety of conditions andthe simple incubation in phosphate buffer and 0.05% Tween was found tobe satisfactory.

Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, et al., MOLECULAR CLONING; A LABORATORY MANUAL, SECOND EDITION(1989); DNA CLONING, VOLUMES I AND II (D. N Glover ed. 1985);OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed, 1984); NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); TRANSCRIPTION ANDTRANSLATION (B. D. Hames & S. J. Higgins eds. 1984); ANIMAL CELL CULTURE(R. I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press,1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); theseries, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFERVECTORS FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol.155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker,eds. (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY(Academic Press, London), Scopes, (1987), PROTEIN PURIFICATION:PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, N.Y.), andHANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES I-IV (D. M. Weir and C. C.Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in thisspecification. All publications, patents, and patent applications citedherein are incorporated by reference.

Examples of the protein that can be used in the present inventioninclude polypeptides with minor amino acid variations from the naturalamino acid sequence of the protein; in particular, conservative aminoacid replacements are contemplated. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids are generally divided intofour families: (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar=glycine, asparagine, glutamine, cystine, serine, threonine,tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimesclassified jointly as aromatic amino acids. For example, it isreasonably predictable that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar conservative replacement of an amino acid with astructurally related amino acid will not have a major effect on thebiological activity. Polypeptide molecules having substantially the sameamino acid sequence as the protein but possessing minor amino acidsubstitutions that do not substantially affect the functional aspectsare within the definition of the protein.

A significant advantage of producing the protein by recombinant DNAtechniques rather than by isolating and purifying a protein from naturalsources is that equivalent quantities of the protein can be produced byusing less starting material than would be required for isolating theprotein from a natural source. Producing the protein by recombinanttechniques also permits the protein to be isolated in the absence ofsome molecules normally present in cells. Indeed, protein compositionsentirely free of any trace of human protein contaminants can readily beproduced because the only human protein produced by the recombinantnon-human host is the recombinant protein at issue. Potential viralagents from natural sources and viral components pathogenic to humansare also avoided.

The term “recombinant polynucleotide” as used herein intends apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of a polynucleotide with which it is associated innature, (2) is linked to a polynucleotide other than that to which it islinked in nature, or (3) does not occur in nature.

The term “polynucleotide” as used herein refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, this term includes double- and single-stranded DNAand RNA. It also includes known types of modifications, for example,labels which are known in the art, methylation, “caps”, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example proteins (including for e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide.

A “replicon” is any genetic element, e.g., a plasmid, a chromosome, avirus, a cosmid, etc. that behaves as an autonomous unit ofpolynucleotide replication within a cell; i.e., capable of replicationunder its own control. This may include selectable markers.

A “vector” is a replicon in which another polynucleotide segment isattached, so as to bring about the replication and/or expression of theattached segment.

“Control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequence” is intended to include, at a minimum, all components whosepresence is necessary for expression, and may also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.

An “open reading frame” (ORF) is a region of a polynucleotide sequencewhich encodes a polypeptide; this region may represent a portion of acoding sequence or a total coding sequence.

A “coding sequence” is a polynucleotide sequence which is translatedinto a polypeptide, usually via mRNA, when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a translation start codon at the 5′-terminus and atranslation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to, cDNA, and recombinant polynucleotidesequences.

“PCR” refers to the technique of polymerase chain reaction as describedin Saiki, et al., Nature 324:163 (1986); and Scharf et al., Science(1986) 233:1076-1078; and U.S. Pat. No. 4,683,195; and U.S. Pat. No.4,683,202. As used herein, x is “heterologous” with respect to y if x isnot naturally associated with y in the identical manner; i.e., x is notassociated with y in nature or x is not associated with y in the samemanner as is found in nature.

“Homology” refers to the degree of similarity between x and y. Thecorrespondence between the sequence from one form to another can bedetermined by techniques known in the art. For example, they can bedetermined by a direct comparison of the sequence information of thepolynucleotide. Alternatively, homology can be determined byhybridization of the polynucleotides under conditions which form stableduplexes between homologous regions (for example, those which would beused prior to S₁ digestion), followed by digestion with single-strandedspecific nuclease(s), followed by size determination of the digestedfragments.

As used herein, the term “polypeptide” refers to a polymer of aminoacids and does not refer to a specific length of the product; thus,peptides, oligopeptides, and proteins are included within the definitionof polypeptide. This term also does not refer to or exclude postexpression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, polypeptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

A polypeptide or amino acid sequence “derived from” a designated nucleicacid sequence refers to a polypeptide having an amino acid sequenceidentical to that of a polypeptide encoded in the sequence, or a portionthereof wherein the portion consists of at least 3-5 amino acids, andmore preferably at least 8-10 amino acids, and even more preferably atleast 11-15 amino acids, or which is immunologically identifiable with apolypeptide encoded in the sequence. This terminology also includes apolypeptide expressed from a designated nucleic acid sequence.

The protein may be used for producing antibodies, either monoclonal orpolyclonal, specific to the protein. The methods for producing theseantibodies are known in the art.

“Recombinant host cells”, “host cells,” “cells,” “cell cultures,” andother such terms denote, for example, microorganisms, insect cells, andmammalian cells, that can be, or have been, used as recipients forrecombinant vector or other transfer DNA, and include the progeny of theoriginal cell which has been transformed. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.Examples for mammalian host cells include Chinese hamster ovary (CHO)and monkey kidney (COS) cells.

Specifically, as used herein, “cell line,” refers to a population ofcells capable of continuous or prolonged growth and division in vitro.Often, cell lines are clonal populations derived from a singleprogenitor cell. It is further known in the art that spontaneous orinduced changes can occur in karyotype during storage or transfer ofsuch clonal populations. Therefore, cells derived from the cell linereferred to may not be precisely identical to the ancestral cells orcultures, and the cell line referred to includes such variants. The term“cell lines” also includes immortalized cells. Preferably, cell linesinclude nonhybrid cell lines or hybridomas to only two cell types.

As used herein, the term “microorganism” includes prokaryotic andeukaryotic microbial species such as bacteria and fungi, the latterincluding yeast and filamentous fungi.

“Transformation”, as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for the insertion, for example, direct uptake, transduction,f-mating or electroporation. The exogenous polynucleotide may bemaintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host genome.

By “genomic” is meant a collection or library of DNA molecules which arederived from restriction fragments that have been cloned in vectors.This may include all or part of the genetic material of an organism.

By “cDNA” is meant a complimentary mRNA sequence that hybridizes to acomplimentary strand of mRNA.

By “purified” and “isolated” is meant, when referring to a polypeptideor nucleotide sequence, that the indicated molecule is present in thesubstantial absence of other biological macromolecules of the same type.The term “purified” as used herein preferably means at least 75% byweight, more preferably at least 85% by weight, more preferably still atleast 95% by weight, and most preferably at least 98% by weight, ofbiological macromolecules of the same type present (but water, buffers,and other small molecules, especially molecules having a molecularweight of less than 1000, can be present).

Expression Systems

Once the appropriate coding sequence is isolated, it can be expressed ina variety of different expression systems; for example those used withmammalian cells, baculoviruses, bacteria, and yeast.

i. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoteris any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of a coding sequence (e.g.structural gene) into mRNA. A promoter will have a transcriptioninitiating region, which is usually placed proximal to the 5′ end of thecoding sequence, and a TATA box, usually located 25-30 base pairs (bp)upstream of the transcription initiation site. The TATA box is thoughtto direct RNA polymerase II to begin RNA synthesis at the correct site.A mammalian promoter will also contain an upstream promoter element,usually located within 100 to 200 bp upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation [Sambrook et al. (1989)“Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: ALaboratory Manual, 2nd ed.].

Mammalian viral genes are often highly expressed and have a broad hostrange; therefore sequences encoding mammalian viral genes provideparticularly useful promoter sequences. Examples include the SV40 earlypromoter, mouse mammary tumor virus LTR promoter, adenovirus major latepromoter (Ad MLP), and herpes simplex virus promoter. In addition,sequences derived from non-viral genes, such as the murinemetallotheionein gene, also provide useful promoter sequences.Expression may be either constitutive or regulated (inducible),depending on the promoter can be induced with glucocorticoid inhormone-responsive cells.

The presence of an enhancer element (enhancer), combined with thepromoter elements described above, will usually increase expressionlevels. An enhancer is a regulatory DNA sequence that can stimulatetranscription up to 1000-fold when linked to homologous or heterologouspromoters, with synthesis beginning at the normal RNA start site.Enhancers are also active when they are placed upstream or downstreamfrom the transcription initiation site, in either normal or flippedorientation, or at a distance of more than 1000 nucleotides from thepromoter [Maniatis et al. (1987) Science 236:1237; Alberta et al. (1989)Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived fromviruses may be particularly useful, because they usually have a broaderhost range. Examples include the SV40 early gene enhancer [Dijkema et al(1985) EMBO J. 4:761] and the enhancer/promoters derived from the longterminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b)Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus [Boshartet al. (1985) Cell 41:521]. Additionally, some enhancers are regulatableand become active only in the presence of an inducer, such as a hormoneor metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215;Maniatis et al. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. Apromoter sequence may be directly linked with the DNA molecule, in whichcase the first amino acid at the N-terminus of the recombinant proteinwill always be a methionine, which is encoded by the ATG start codon. Ifdesired, the N-terminus may be cleaved from the protein by in vitroincubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provides forsecretion of the foreign protein in mammalian cells. Preferably, thereare processing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell. Theadenovirus triparite leader is an example of a leader sequence thatprovides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-transcriptional cleavage and polyadenylation[Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988)“Termination and 3′ end processing of eukaryotic RNA. In Transcriptionand splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) TrendsBiochem. Sci. 14:105]. These sequences direct the 5 transcription of anmRNA which can be translated into the polypeptide encoded by the DNA.Examples of transcription terminater/polyadenylation signals includethose derived from SV40 [Sambrook et al (1989) “Expression of clonedgenes in cultured mammalian cells.” In Molecular Cloning: A LaboratoryManual].

Some genes may be expressed more efficiently when introns (also calledintervening sequences) are present. Several cDNAs, however, have beenefficiently expressed from vectors that lack splicing signals (alsocalled splice donor and acceptor sites) [see e.g., Gothing and Sambrook(1981) Nature 293:620]. Introns are intervening noncoding sequenceswithin a coding sequence that contain splice donor and acceptor sites.They are removed by a process called “splicing,” followingpolyadenylation of the primary transcript [Nevins (1983) Annu. Rev.Biochem. 52:441; Green (1986) Annu. Rev. Genet. 20:671; Padgett et al.(1986) Annu. Rev. Biochem. 55:1119; Krainer and Maniatis (1988) “RNAsplicing.” In Transcription and splicing (ed. B. D. Hames and D. M.Glover)].

Usually, the above described components, comprising a promoter,polyadenylation signal, and transcription termination sequence are puttogether into expression constructs. Enhancers, introns with functionalsplice donor and acceptor sites, and leader sequences may also beincluded in an expression construct, if desired. Expression constructsare often maintained in a replicon, such as an extrachromosomal element(e.g., plasmids) capable of stable maintenance in a host, such asmammalian cells or bacteria. Mammalian replication systems include thosederived from animal viruses, which require trans-acting factors toreplicate. For example, plasmids containing the replication systems ofpapovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] orpolyomavirus, replicate to extremely high copy number in the presence ofthe appropriate viral T antigen. Additional examples of mammalianreplicons include those derived from bovine papillomavirus andEpstein-Barr virus. Additionally, the replicon may have two replicatonsystems, thus allowing it to be maintained, for example, in mammaliancells for expression and in a procaryotic host for cloning andamplification. Examples of such mammalian-bacteria shuttle vectorsinclude pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946 and pHEBO(Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to betransformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to, Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g.,Hep G2), and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into asuitable insect expression vector, and is operably linked to the controlelements within that vector. Vector construction employs techniqueswhich are known in the art.

Generally, the components of the expression system. include a transfervector, usually a bacterial plasmid, which contains both a fragment ofthe baculovirus genome, and a convenient restriction site for insertionof the heterologous gene or genes to be expressed; a wild typebaculovirus with a sequence homologous to the baculovirus-specificfragment in the transfer vector (this allows for the homologousrecombination of the heterologous gene in to the baculovirus genome);and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfervector, the vector and the wild type viral genome are transfected intoan insect host cell where the vector and viral genome are allowed torecombine. The packaged recombinant virus is expressed and recombinantplaques are identified and purified. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, inter alia, Invitrogen, San Diego Calif. (^(□)MaBac^(□)kit). These techniques are generally known to those skilled in the artand fully described in Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987) (hereinafter ^(□)Summers andSmith^(□)).

Prior to inserting the DNA sequence encoding the protein into thebaculovirus genome, the above described components, comprising apromoter, leader (if desired), coding sequence of interest, andtranscription termination sequence, are usually assembled into anintermediate transplacement construct (transfer vector). This constructmay contain a single gene and operably linked regulatory elements;multiple genes, each with its owned set of operably linked regulatoryelements; or multiple genes, regulated by the same set of regulatoryelements. Intermediate transplacement constructs are often maintained ina replicon, such as an extrachromosomal element (e.g., plasmids) capableof stable maintenance in a host, such as a bacterium. The replicon willhave a replication system, thus allowing it to be maintained in asuitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 basepairs downstreamfrom the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal(Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a procaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence (e.g. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region usually includes an RNA polymerase binding site and atranscription initiation site. A baculovirus transfer vector may alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in a viralinfection cycle, provide particularly useful promoter sequences.Examples include sequences derived from the gene encoding the viralpolyhedron protein, Friesen et al., (1986) “The Regulation ofBaculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses(ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the geneencoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes forsecreted insect or baculovirus proteins, such as the baculoviruspolyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively,since the signals for mammalian cell posttranslational modifications(such as signal peptide cleavage, proteolytic cleavage, andphosphorylation) appear to be recognized by insect cells, and thesignals required for secretion and nuclear accumulation also appear tobe conserved between the invertebrate cells and vertebrate cells,leaders of non-insect origin, such as those derived from genes encodinghuman α-interferon, Maeda et al., (1985), Nature 315:592; humangastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell.Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci.USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; andhuman glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also beused to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressedintracellularly or, if it is expressed with the proper regulatorysequences, it can be secreted. Good intracellular expression of nonfusedforeign proteins usually requires heterologous genes that ideally have ashort leader sequence containing suitable translation initiation signalspreceding an ATG start signal. If desired, methionine at the N-terminusmay be cleaved from the mature protein by in vitro incubation withcyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are notnaturally secreted can be secreted from the insect cell by creatingchimeric DNA molecules that encode a fusion protein comprised of aleader sequence fragment that provides for secretion of the foreignprotein in insects. The leader sequence fragment usually encodes asignal peptide comprised of hydrophobic amino acids which direct thetranslocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding theexpression product precursor of the protein, an insect cell host isco-transformed with the heterologous DNA of the transfer vector and thegenomic DNA of wild type baculovirus—usually by co-transfection. Thepromoter and transcription termination sequence of the construct willusually comprise a 2-5 kb section of the baculovirus genome. Methods forintroducing heterologous DNA into the desired site in the baculovirusvirus are known in the art. (See Summers and Smith supra; Ju et al.(1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow andSummers (1989)). For example, the insertion can be into a gene such asthe polyhedrin gene, by homologous double crossover recombination;insertion can also be into a restriction enzyme site engineered into thedesired baculovirus gene. Miller et al., (1989), Bioessays 4:91. The DNAsequence, when cloned in place of the polyhedrin gene in the expressionvector, is flanked both 5′ and 3′ by polyhedrin-specific sequences andis positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packagedinto an infectious recombinant baculovirus. Homologous recombinationoccurs at low frequency (between about 1% and about 5%); thus, themajority of the virus produced after cotransfection is still wild-typevirus. Therefore, a method is necessary to identify recombinant viruses.An advantage of the expression system is a visual screen allowingrecombinant viruses to be distinguished. The polyhedrin protein, whichis produced by the native virus, is produced at very high levels in thenuclei of infected cells at late times after viral infection.Accumulated polyhedrin protein forms occlusion bodies that also containembedded particles. These occlusion bodies, up to 15 μm in size, arehighly retractile, giving them a bright shiny appearance that is readilyvisualized under the light microscope. Cells infected with recombinantviruses lack occlusion bodies. To distinguish recombinant virus fromwild-type virus, the transfection supernatant is plagued onto amonolayer of insect cells by techniques known to those skilled in theart. Namely, the plaques are screened under the light microscopy for thepresence (indicative of wild-type virus) or absence (indicative ofrecombinant virus) of occlusion bodies. ^(□)Current Protocols inMicrobiology^(□) Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990);Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia: Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni (PCT Pub. No. WO 89/046699; Carbonell etal., (1985) J. Virol, 56:153; Wright (1986) Nature 321:718; Smith etal., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al.(1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both directand fusion expression of heterologous polypeptides in abaculovirus/expression system; cell culture technology is generallyknown to those skilled in the art. See, e.g., Summers and Smith supra.

The modified insect cells may then be grog in an appropriate nutrientmedium, which allows for stable maintenance of the plasmid(s) present inthe modified insect host. Where the expression product gene is underinducible control, the host may be grown to high density, and expressioninduced. Alternatively, where expression is constitutive, the productwill be continuously expressed into the medium and the nutrient mediummust be continuously circulated, while removing the product of interestand augmenting depleted nutrients. The product may be purified by suchtechniques as chromatography, e.g., HPLC, affinity chromatography, ionexchange chromatography, etc.; electrophoresis; density gradientcentrifugation; solvent extraction, or the like. As appropriate, theproduct may be further purified, as required, so as to removesubstantially any insect proteins which are also secreted in the mediumor result from lysis of insect cells, so as to provide a product whichis at least substantially free of host debris, e.g., proteins, lipidsand polysaccharides.

In order to obtain protein expression, recombinant host cells derivedfrom the transformants are incubated under conditions which allowexpression of the recombinant protein encoding sequence. Theseconditions will vary, dependent upon the host cell selected. However,the conditions are readily ascertainable to those of ordinary skill inthe art, based upon what is known in the art.

iii. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterialpromoter is any DNA sequence capable of binding bacterial RNA polymeraseand initiating the downstream (3″) transcription of a coding sequence(e.g. structural gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5′ end of thecoding sequence. This transcription initiation region usually includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter may also have a second domain called an operator,that may overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein may bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression may occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation may be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5′) to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in Escherichia coli(E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulatedexpression may therefore be either positive or negative, thereby eitherenhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal. (1977) Nature 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al.(1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPO Publ. Nos.036 776 and 121 775]. The g-laotamase (bla) promoter system [Weissmann(1981) “The cloning of interferon and other mistakes.” In Interferon 3(ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981)Nature Z29:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems alsoprovide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. Forexample, the tac promoter is a hybrid trp-lac promoter comprised of bothtrp promoter and lac operon sequences that is regulated by the lacrepressor [Amann et al. (1983) Gene 2:167; de Boer et al. (1983) Proc.Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can includenaturally occurring promoters of non-bacterial origin that have theability to bind bacterial RNA polymerase and initiate transcription. Anaturally occurring promoter of non-bacterial origin can also be coupledwith a compatible RNA polymerase to produce high levels of expression ofsome genes in prokaryotes. The bacteriophase T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al. (1986)J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci.82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EPO Publ. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine at al. (1975) Nature 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences inmessenger RNA.” In Biological Regulation and Development: GeneExpression (ed. R. F. Goldberger)]. To express eukaryotic genes andprokaryotic genes with weak ribosome-binding site [Sambrook et al.(1989) “Expression of cloned genes in Escherichia coli.” In MolecularCloning: A Laboratory Manual].

A DNA molecule may be expressed intracellularly. A promoter sequence maybe directly linked with the DNA molecule, in which case the first aminoacid at the N-terminus will always be a methionine, which is encoded bythe ATG start codon. If desired, methionine at the N-terminus may becleaved from the protein by in vitro incubation with cyanogen bromide orby either in vivo on in vitro incubation with a bacterial methionineN-terminal peptidase (EPO Publ. No. 219 237).

Fusion proteins provide an alternative to direct expression. Usually, aDNA sequence encoding the N-terminal portion of an endogenous bacterialprotein, or other stable protein, is fused to the 5′ end of heterologouscoding sequences. Upon expression, this construct will provide a fusionof the two amino acid sequences. For example,-the bacteriophage lambdacell gene can be linked at the 5′ terminus of a foreign gene andexpressed in bacteria. The resulting fusion protein preferably retains asite for a processing enzyme (factor Xa) to cleave the bacteriophageprotein from the foreign gene [Nagai et al. (1984) Nature 309:810].Fusion proteins can also be made with sequences from the lacZ [Jia etal. (1987) Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93;Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EPO Publ. No.324 647] genes. The DNA sequence at the junction of the two amino acidsequences may or may not encode a cleavable site. Another example is aubiquitin fusion protein. Such a fusion protein is made with theubiquitin region that preferably retains a site for a processing enzyme(e.g. ubiquitin specific processing-protease) to cleave the ubiquitinfrom the foreign protein. Through this method, native foreign proteincan be isolated [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secreted from the cell bycreating chimeric DNA molecules that encode a fusion protein comprisedof a signal peptide sequence fragment that provides for secretion of theforeign protein in bacteria [U.S. Pat. No. 4,336,336]. The signalsequence fragment usually encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic spece, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).Preferably there are processing sites, which can be cleaved either invivo or in vitro encoded between the signal peptide fragment and theforeign gene.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA) [Masui et al. (1983), in: Experimental Manipulation of GeneExpression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. colialkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc.Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequenceof the alpha-amylase gene from various Bacillus strains can be used tosecrete heterologous proteins from B. subtilis [Palva et al. (1982)Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. No. 244 042].

Usually, transcription termination sequences recognized by bacteria areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Transcription termination sequencesfrequently include DNA sequences of about 50 nucleotides capable offorming stem loop structures that aid in terminating transcription.Examples include transcription termination sequences derived from geneswith strong promoters, such as the trp gene in E. coli as well as otherbiosynthetic genes.

Usually, the above described components, comprising a promoter, signalsequence (if desired), coding sequence of interest, and transcriptiontermination sequence, are put, together into expression constructs.Expression constructs are often maintained in a replicon, such as anextrachromosomal element (e.g., plasmids) capable of stable maintenancein a host, such as bacteria. The replicon will have a replicationsystem, thus allowing it to be maintained in a procaryotic host eitherfor expression or for cloning and amplification. In addition, areplicoin may be either a high or low copy number plasmid. A high copynumber plasmid will generally have a copy number ranging from about 5 toabout 200, and usually about 10 to about 150. A host containing a highcopy number plasmid will preferably contain at least about 10, and morepreferably at least about 20 plasmids. Either a high or low copy numbervector may be selected, depending upon the effect of the vector and theforeign protein on the host.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to the bacterial chromosomethat allows the vector to integrate. Integrations appear to result fromrecombinations between homologous DNA in the vector and the bactedrialchromosome. For example, integrating vectors constructed with DNA fromvarious Bacillus strains integrate into the Bacillus chromosome (EPOPubl. No. 127 328). Integrating vectors may also be comprised ofbacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and may include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev.Microbiol. 32:469]. Selectable markers may also include biosyntheticgenes, such as those in the histidine, tryptophan, and leucinebiosynthetic pathways.

Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are usuallycomprised of a selectable market that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomalreplicons or integrating vectors, have been developed for transformationinto many bacteria. For example, expression vectors have been developedfor, inter alia, the following bacteria: Bacillus subtilis [Palva et al.(1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. Nos. 036 259 and063 953; PCT Publ. No. WO 84/04541], Escherichia coli [Shimatake et al.(1981) Nature 292:128; al. Amann et al. (1985) Gene 40:183; Studier etal. (1986) J. Mol. Biol. 189:113; EPO Publ. Nos. 036 776, 136 829 and136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ.Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl.Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No.4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and usually include either the transformation of bacteriatreated pith CaCl₂ or other agents, such as divalent cations and DMSO.DNA can also be introduced into bacterial cells by electroporation.Transformation procedures usually vary with the bacterial species to betransformed. See e.g., [Masson et al. (1989) FEMS Microbiol. Lett.60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EPOPubl. Nos. 036 259 and 063 953; PCT Publ. No. WO 84/04541, Bacillus],[Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990)J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl.Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127;Kushner (1978) ^(□)An improved method for transformation of Escherichiacoli with ColE1-derived plasmids. In Genetic Engineering: Proceedings ofthe International Symposium on Genetic Engineering (eds. H. W. Boyer andS. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988)Biochim. Biophys. Acta 949:318; Escherichia], (Chassy et al. (1987) FEMSMicrobiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal.Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol.Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol.144:698; Harlander (1987) “Transformation o Streptococcus lactis byelectroporation, in: Streptococcal Genetics (ed. J. Ferretti and R.Curtiss III); Perry et al. (1981) Infec. Immun. 32:1295; Powell at al.(1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4thEvr. Cong. Biotechnolgy 1:412, Streptococcus].

iv. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in theart. A yeast promoter is any DNA sequence capable of binding yeast RNApolymerase and initiating the downstream (3′) transcription of a codingsequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regionusually includes an RNA polymerase binding site (the “TATA Box”) and atranscription initiation site. A yeast promoter may also have a seconddomain called an upstream activator sequence (UAS), which, if present,is usually distal to the structural gene. The UAS permits regulated(inducible) expression. Constitutive expression occurs in the absence ofa UAS. Regulated expression may be either positive or negative, therebyeither enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway,therefore sequences encoding enzymes in the metabolic pathway provideparticularly useful promoter sequences. Examples include alcoholdehydrogenase (ADH) (EPO Publ. No. 284 044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK) (EPO Publ. No. 329 203). The yeastPHO5 gene, encoding acid phosphatase, also provides useful promotersequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].

In addition, synthetic promoters which do not occur in nature alsofunction as yeast promoters. For example, UAS sequences of one yeastpromoter may be joined with the transcription activation region ofanother yeast promoter, creating a synthetic hybrid promoter. Examplesof such hybrid promoters include the ADH regulatory sequence linked tothe GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and4,880,734). Other examples of hybrid promoters include promoters whichconsist of the regulatory sequences of either the ADH2, GAL4, GAL10, ORPHO5 genes, combined with the transcriptional activation region of aglycolytic enzyme gene such as GAP or PyK (EPO Publ. No. 164 556).Furthermore, a yeast promoter can include naturally occurring promotersof non-yeast origin that have the ability to bind yeast RNA polymeraseand initiate transcription. Examples of such promoters include, interalia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoffet al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. TopicsMicrobiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression ofBacterial Antibiotic Resistance Genes i the Yeast Saccharomycescerevisiae,” in: Plasmids of Medical, Environmental and CommercialImportance (eds. K>N>Timmis and A. Puhler); Mercerau-Puigalon et al.(1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].

A DNA molecule may be expressed intracellularly in yeast. A promotersequence may be directly linked with the DNA molecule, in which case thefirst amino acid at the N-terminus of the recombinant protein willalways be a methionine, which is encoded by the ATG start codon. Ifdesired, methionine at the N-terminus may be cleaved from the protein byin vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, aswell as in mammalian, baculovirus, and bacterial expression systems.Usually, a DNA sequence encoding the N-terminal portion of an endogenousyeast protein, or other stable protein, is fused to the 5′ end ofheterologous coding sequences. Upon expression, this construct willprovide a fusion of the two amino acid sequences. For example, the yeastor human superoxide dismutase (SOD) gene, can be linked at the 5′terminus of a foreign gene and expressed in yeast. The DNA sequence atthe junction of the two amino acid sequences may or may not encode acleavable site. See e.g., EPO Publ. No. 196 056. Another example is aubiquitin fusion protein. Such a fusion protein is made with theubiquitin region that preferably retains a site for a processing enzyme(e.g. ubiquitin-specific processing protease) to cleave the ubiquitinfrom the foreign protein. Through this method, therefore, native foreignprotein can be isolated (see, e.g., PCT Publ. No. WO 88/024066).

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provide forsecretion in yeast of the foreign protein. Preferably, there areprocessing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the yeast invertase gene (EPO Publ. No.012 873; JPO Publ. No. 62,096,086) and the A-factor gene (U.S. Pat. No.4,588,684). Alternatively, leaders of non-yeast origin, such as aninterferon leader, exist that also provide for secretion in yeast (EPOPubl. No. 060 057).

A preferred class of secretion leaders are those that employ a fragmentof the yeast alpha-factor gene, which contains both a ^(□)pre^(□) signalsequence, and a ^(□)pro^(□) region. The types of alpha-factor fragmentsthat can be employed include the full-length pre-pro alpha factor leader(about 83 amino acid residues) as well as truncated alpha-factor leaders(usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos.4,586,083 and 4,870,008; EPO Publ. No. 324 274). Additional leadersemploying an alpha-factor leader fragment that provide a for secretioninclude hybrid alpha-factor leaders made with a presequence of a firstyeast, but a pro-region from a second yeast alphafactor. (See e.g., PCTPubl. No. WO 89/02463.)

Usually, transcription termination sequences recognized by yeast areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Examples of transcription terminatorsequence and other yeast-recognized termination sequences, such as thosecoding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader(if desired), coding sequence of interest, and transcription terminationsequence, are put together into expression constructs. Expressionconstructs are often maintained in a replicon, such as anextrachromosomal element (e.g., plasmids) capable of stable maintenancein a host, such as yeast or bacteria. The replicon may have tworeplication systems, thus allowing it to be maintained, for example, inyeast for expression and in a procaryotic host for cloning andamplification. Examples of such yeast-bacteria shuttle vectors includeYEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984)Proc. Natl. Acad. Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al.(1982) J. Mol. Biol, 158:157]. In addition, a replicon may be either ahigh or low copy number plasmid. A high copy number plasmid willgenerally have a copy number ranging from about 5 to about 200, andusually about 10 to about 150. A host containing a high copy numberplasmid will preferably have at least about 10, and more preferably atleast about 20. Enter a high or low copy number vector may be selected,depending upon the effect of the vector and the foreign protein on thehost. See e.g., Brake et al., spura.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol.101:228-245]. An integrating vector may be directed to a specific locusin yeast by selecting the appropriate homologous sequence for inclusionin the vector. See Orr-Weaver et al., supra. One or more expressionconstruct may integrate, possibly affecting levels of recombinantprotein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA80:6750]. The chromosomal sequences included in the vector can occureither as a single segment in the vector, which results in theintegration of the entire vector, or two segments homologous to adjacentsegments in the chromosome and flanking the expression construct in thevector, which can result in the stable integration of only theexpression construct.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of yeast strainsthat have been transformed. Selectable markers may include biosyntheticgenes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2,TRP1, and ALG7, and the G418 resistance gene, which confer resistance inyeast cells to tunicamycin and G418, respectively. In addition, asuitable selectable marker may also provide yeast with the ability togrow in the presence of toxic compounds, such as metal. For example, thepresence of CUP1 allows yeast to grow in the presence of copper ions[Butt et al. (1987) Microbiol. Rev. 51:351].

Alternatively, some of the above described components can be puttogether into transformation vectors. Transformation vectors are usuallycomprised of a selectable marker that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal repliconsor integrating vectors, have been developed for transformation into manyyeasts. For example, expression vectors have been developed for, interalia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol.Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. BasicMicrobiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen.Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302],Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165],Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol.154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichiaguillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichiapastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos.4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978)Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol.153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet.10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known inthe art, and usually include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationprocedures usually vary with the yeast species to be transformed. Seee.g., [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985)J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen.Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302;Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt etal. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990)Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell.Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat.Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen e al. (1978) Proc. Natl.Acad. Sci. USA 75;1929; Ito et al. (1983) J. Bacteriol. 153:163Saccharomyces]; [Beach and Nurse (1981) Nature 300:706;Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39;Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

Nucleic Acid Assays

Polynucleotide probes of approximately 8 nucleotides or more can beprepared which hybridize with the positive strand(s) of the RNA or itscomplement, as well as to cDNAs. These polynucleotides serve as probesfor the detection, isolation and/or labeling of polynucleotides whichcontain nucleotide sequences, and/or as primers for the transcriptionand/or replication of the targeted sequences. Each probe contains atargeting polynucleotide sequence, which is comprised of nucleotideswhich are complementary to a target nucleotide sequence; the sequence isof sufficient length and complementarily with the sequence to form aduplex which has sufficient stability for the purpose intended. Forexample, if the purpose is the isolation, via immobilization, of ananalyte containing a target sequence, the probes will contain apolynucleotide region which is of sufficient length and complementarilyto the targeted sequence to afford sufficient duplex stability toimmobilize the analyte on a solid surface under the isolationconditions. For example, also, if the polynucleotide probes are to serveas primers for the transcription and/or replication of target sequences,the probes will contain a polynucleotide region of sufficient length andcomplementarily to the targeted sequence to allow for replication. Forexample, also,.if the polynucleotide probes are to be used as labelprobes, or are to bind to multimers, the targeting polynucleotide regionwould be of sufficient length and complementarily to form stable hybridduplex structures with the label probes and/or multimers to allowdetection of the duplex. The probes may contain a minimum of about 4contiguous nucleotides which are complementary to the targeted sequence;usually the oligomers will contain a minimum of about 8 continuousnucleotides which are complementary to the targeted sequence, andpreferably will contain a minimum of about 14 contiguous nucleotideswhich are complementary to the targeted sequence.

The probes, however, need not consist only of the sequence which iscomplementary to the targeted sequence. They may contain additionalnucleotide sequences or other moieties. For example, if the probes areto be used as primers for the amplification of sequences via PCR, theymay contain sequences which, when in duplex, form restriction enzymesites which facilitate the cloning of the amplified sequences. Forexample, also, if the probes are to be used as “capture probes” inhybridization assays, they will be coupled to a “binding partners” asdefined above. Preparation of the probes is by means known in the art,including, for example, by methods which include excision, transcriptionor chemical synthesis.

Immunodiagnostic Assays

Antigens can be used in immunoassays to detect antibody levels (orconversely antibodies can be used to detect antigen levels) andcorrelation can be made with disease. Immunoassays based on welldefined, recombinant antigens can be developed to replace the invasivediagnostics methods that are used today. Antibodies to proteins withinbiological samples, including for S example, blood or serum samples, canbe detected. Design of the immunoassays is subject to a great deal ofvariation, and a variety of these are known in the art. Protocols forthe immunoassay may be based, for example, upon competition, or directreaction, or sandwich type assays. Protocols may also, for example, usesolid supports, or may be by immunoprecipitation. Most assays involvethe use of labeled antibody or polypeptide; the labels may be, forexample, fluorescent, chemiluminescent, radioactive, or dye molecules.Assays which amplify the signals from the probe are also known; examplesof which are assays which utilize biotin and avidin, and enzyme-labeledand mediated immunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriate labeledreagents are constructed by packaging the appropriate materials,including the compositions of the invention, in suitable containers,along with the remaining reagents and materials (for example, suitablebuffers, salt solutions, etc.) required for the conduct of the assay, aswell as suitable set of assay instructions.

Vaccines

Vaccines may either be prophylactic (to prevent infection) ortherapeutic (to treat disease after infection).

Such vaccines comprise antigen or antigens, usually in combination with“pharmaceutically acceptable carriers,” which include any carrier thatdoes not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes), and inactive virus particles. Such carriers are well knownto those of ordinary skill in the art. Additionally, these carriers mayfunction as immunostimulating agents (“adjuvants”). Furthermore, theantigen may be conjugated to a bacterial toxoid, such as a toxoid fromdiphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: (1) aluminum salts (alum), such as aluminumhydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-wateremulsion formulations (with or without other specific immunostimulatingagents such as muramyl peptides (see below) or bacterial cell wallcomponents), such as for example (a) MF59 (PCT Publ. No. WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE (see below), although not required)formulated into submicron particles using a microfluidizer such as Model110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, andthr-MDP (see below) either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion, and (c) Ribi™adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mon.) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentsfrom the group consisting of monophosphorylipid A (MPL), trehalosedimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS(Detox™); (3) saponin adjuvants, such as Stimulon™ (CambridgeBioscience, Worcester, Mass.) may be used or particles generatedtherefrom such as ISCOMs (immunostimulating complexes); (4) CompleteFreunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5)cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colonystimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6)other substances that act as immunostimulating agents to enhance theeffectiveness of the composition. Alum and MF59 are preferred.

As mentioned above, muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

The immunogenic compositions (e.g., the antigen, pharmaceuticallyacceptable carrier, and adjuvant) typically will contain diluents, suchas water, saline, glycerol, ethanol, etc. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect, as discussed above underpharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of the antigenic polypeptides, as well as any other ofthe above-mentioned components, as needed. By “immunologically effectiveamount”, it is meant that the administration of that amount to anindividual, either in a single dose or as part of a series, is effectivefor treatment or prevention. This amount varies depending upon thehealth and physical condition of the individual to be treated, thetaxonomic group of individual to be treated (e.g., nonhuman primate,primate, etc.), the capacity of the individual's immune system tosynthesize antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials.

The immunogenic compositions are conventionally administeredparenterally, e.g., by injection, either subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications. Dosage treatment may be a single doseschedule or a multiple dose schedule. The vaccine may be administered inconjunction with other immunoregulatory agents.

EXAMPLES

The examples presented herein are provided as a further guide to thepractitioner of ordinary skill in the art and are not to be construed aslimiting the invention in any way.

Example 1 Recombinant Antigen Preparation

The ORF3 sequence of pCT cloned from a serotype D isolate of C.trachomatis (Comanducci et al. 1990) was sub-cloned in E. coli strainBL21, using the plasmid expression vector pT7-7 (Tabor et al., Proc.Natl. Acad. Sci., USA, 1985). In this system the expression of ORF3 isunder the control of an IPTG-inducible promoter and yields theunmodified pgp3 amino acid sequence. The recombinant pgp3 protein waspurified from E. coli cell extracts by ion-exchange columnchromatography on mono-Q prepacked columns (Pharmacia), using a NaClelution gradient in piperazine-HCl buffer (Comanducci et al., manuscriptin preparation). The final product was >90% pure pgp3 antigen, accordingto electrophoretic and immunoblot analyses which were done using thepreviously described rabbit anti serum raised against the 39 kDa fusionprotein (Comanducci et al. 1993).

Example 2 Western Blot Analysis and Micro-Immunofluorescence

Western blot analysis and micro-immunofluorescence (MIF) were performedaccording standard procedures as previously described (Comanducci etal., 1993). Human sera were titrated by single-antigen MIF using sucrosegradient purified EBs of C. trachomatis L2434-Bu, C. psittaci 6BC andA22, C. pneumoniae IOL-207.

Example 3 Enzyme Linked Immunoassay

Rabbit sera raised against the pgp3 polypeptide (either against the39-kDa fusion S protein or the 28-kDa recombinant pgp3), andspecifically recognizing a 28-kDa band in Western blots of total EBprotein, were initially used for setting up the assay. Several amounts(500 ng down to 50 ng) of purified antigen in each well were testedagainst reference sera and the amount of 200 ng/well was eventuallychosen as a standard assay condition.

A panel of 21 human sera was used to test the ELISA performance withclinical samples. The appropriate amount (100-500 ng per well) ofpurified pgp3 antigen was diluted in 100 ul of coating buffer (PBS pH8.0, 0.05% Tween 20, 0.02% NaN3) and adsorbed onto plastic wells ofmicrotiter plates (NUNC), for 2 hrs at 37° C. and then overnight at 4°C. The wells then were washed several times with the PBS-Tween buffer,then saturated with 200 microliters of 2.7% Polyvinylpirrolidone inwater for 2 hr and washed again. Serum samples were serially diluted in100 microliters of PBS, and put in contact with the plastic adsorbedantigen for 2 hrs. After adequate washings, bound serum antibodies weremeasured by incubating with alkaline phosphatase labelled anti-rabbit oranti-human IgG antibody (Cappel), followed by a colorimetric reactionusing ELISA substrate and buffer (Sclavo) as recommended by themanufacturers. Optical density readings were performed with a titertekMultiscan MCC densitometer.

Two-fold serial dilutions of the sera were tested, in duplicate samples,with the pgp3-ELISA. OD readings were taken after 30 min, 1 hr and afterO/N storage in the cold (−20° C.). All sera gave negative MIF responseto C. psittaci and C. pneumoniae. When tested for C. trachomatisantibodies, using purified L2-serotype EBs, 15 sera were scored as MIFnegative, and. 6 positive, with titers comprised between 1:32 and 1:256.These sera were also tested for their ability to react with the purifiedpgp3 preparation on Western blots: all the MIF negative sera gavenegative results, whereas the MIF positive sera reacted, to variousextents, with the 28-kDa band only. All 15 negative sera gaveconsistently low OD readings (below 0.1), whereas the 6 positive seragave distinctly higher OD readings which proportionally decreased withthe serum concentration in the sample. The [OD v. dilution] curves ofthe positive sera so far tested correlated with the MIF titres. TypicalELISA results are shown in FIG. 1.

Further work on larger numbers of clinical samples is in progress,however the results here presented indicate that this assay can be usedfor sensitive and quantitative detection of anti pgp3 antibodies inpatient sera, and therefore will be an essential tool for studies on thesignificance of the immune response against this newly described antigenin the course of C. trachomatis infections.

1. A method of eliciting an immune response in a subject to arecombinant Chlamydia trachomatis pgp3 protein comprising administeringto the subject an effective amount of an isolated and purifiedrecombinant Chlamydia trachomatis pgp3 protein having its native aminoacid conformation, wherein the recombinant Chlamydia trachomatis pgp3protein is recognized by antibodies against pgp3 conformationalepitopes.
 2. The method of claim 1 wherein the subject is a human.
 3. Amethod of diagnosing an immune response in a subject to a recombinantChlamydia trachomatis pgp3 protein comprising: contacting a sample fromsaid subject with the recombinant Chlamydia trachomatis pgp3 proteinhaving its native amino acid conformation; and detecting the binding ofantibodies to the pgp3 conformational epitopes of said recombinantChlamydia trachomatis pgp3 protein.
 4. The method of claim 3 wherein thesubject is a human.